THEANATOMYOFTHEBACTERIALSURFACE'M.R.J.SALTONDepartmentofBacteriology,U - PDF document

THEANATOMYOFTHEBACTERIALSURFACE'M.R.J.SALTONDepartmentofBacteriology,UniversityofManchester,Manchester,EnglandI.Introduction..........................................................................77II.SurfaceAppendages....................................................................78III.BacterialCapsules......................................................................80A.ChemicalComposition..............................................................80IV.TheCellWall.......................................................................83A.ChemistryofCellWalls.............................................................83V.ProtoplastMembranes.................................................................86A.ChemicalCompositionofMembranes................................................89VI.LocalizationofEnzymesinBacterialCellsandaSummaryoftheComparativeChemicalandBiochemicalAnatomyofGram-positiveandGram-negativeBacteria....................90VII.TheGramStainandtheBacterialSurface................................................91VIII.References............................................................................95I.INTRODUCrIONDespiteitsminutedimensions,thebacterialcellhasbeenquiteamenabletodissectionintoitscomponentpartsand,duringthepasttenorsoyears,agreatdealhasbeenlearnedaboutthechemicalandbiochemicalanatomyofmicro-organisms.Theterm"bacterialanatomy"hasbecomefirmlyentrenchedinourliteratureanditsusagenolongermystifiesoramusesthosebiologistswhoareusedtohandlingthe"coarse"structuresofhigheranimalandplantcells.Thetransitionfromtheolderstudyofbacterialcytologytothemodernfieldofbacterialanatomyhasreallydatedfromtheintroductionoftheelectronmicroscope,theshadow-castingtech-nique,andthedevelopmentofelegantmethodsforslicingupthebacterialcellintoincrediblythinsections.Theseadvanceshaveoccurredatthetimeofatremendousexpansionofourbio-chemicalknowledgeandtheemergenceofawealthofbiochemicalandbiophysicaltechniques1TheOfficeofNavalResearchLectureattheSocietyofAmericanBacteriologistsAnnualMeetingatPhiladelphiain1960withtheabovetitleformedthebasisforthiscontribution.IshouldliketoexpressmysincerethankstotheOfficeofNavalResearchandtheProgramCom-mitteeoftheSocietyformakingthislecturepossible.Thisreviewofarapidlygrowingfieldisnotintendedtobecomprehensivebuthasbeenpreparedinthehopethatitmayfocusattentiononcertainaspectsandperhapsstimulatesomeinterest,toeliminatethelargegapsinourknowl-edge.whichhaveaccompaniedthisperiod.Thusbac-terialanatomyhasbeenconcernednotsolelywiththemicroscopicappearanceofthevariousmorphologicalentitiesofthecell,butalsowiththemoreexcitingandbroaderproblemofinte-gratingboththestructuralandfunctionalpropertiesofthebacterialcell.Thestudentofhumananatomyisonlytoowellawarethatshapeisamostimportantana-tomicalattribute.Thegeneralimportanceofshapeisamplyillustratedbytheanatomicalandaestheticqualitiesofthefemaleform(unfor-tunately,fortechnicalreasons,theslideillus-tratingthispointinthelecturecouldnotbereproducedhere).Sowithbacterialanatomy,theshapeofthecellisofgreatinterestand,asrealizedlongagobyLeeuwenhoek,itisthesurfacestruc-turewhichdeterminesthecharacteristicshapeofthecell.Theelucidationofthesurfacestructureofbacterialcellshasattractedmuchattentioninrecentyearsandfordiversereasons.Thustheimmunologisthasbeeninterestedinthepresenceofspecificantigenicgroupingsonthecellsurface.Thebiochemisthasconcernedhimselfwiththetransportofsubstancesintoandoutofthecellacrossthepermeabilitybarriersandthoseinter-estedinthecontrolofmicroorganismsbyanti-bioticshavebecomeintriguedwiththepossi-bilitiesofkillingbacteriabyinterferingwiththebiosynthesisofsurfacestructures.Theresultsofallthesediverseinvestigationshavebeguntofocusattentiononthebacterialsurfaceandwecannowbegintotalkaboutitsdetailedanatomy.77 M.R.J.SALTONFigure1.Spirillumserpenscellsautolyzedandtrypsindigested,showingflagellaattachedtobasalgranulesinsidethecell(unpublishedelectronmicrographbyR.W.HorneandM.R.J.Salton).X60,000.Thesurfacestructuresofthebacterialcellcanbeconvenientlyclassified(88)assurfaceappend-ages,surfaceadherents,rigidcellwall,andprotoplasmicmembrane.II.SURFACEAPPENDAGESTwomainsurfaceappendagesofbacterialcellsaretheflagella,theorgansoflocomotionofthecell(121),andthefimbriae(27,29).Theflagellahavenowbeenwellcharacterizedchemically(58,121)andthereisgenerallynodifficultyindistinguishingbetweentheappearanceofflagellaandfimbriaeinelectronmicrographsofbacteria,asillustratedinfigures1and2.ThetermfimbriaewasfirstproposedbyDuguidandhiscolleagues(27,29)astheLatinequivalentof"threads,""fibers,"and"filaments"usedinearlierdescriptionsofthesestructures(3,49).ThistermhasgainedwideacceptancealthoughBrinton(13)hassuggestedthewordpili(Latinfor"hair")asasynonym.Theonlyothersurfaceappendagethatshouldbementionedisthaten-counteredintheironbacterium,Gallioniellaferruginea,whichpossesseslongstrandsofmaterialthatareobviouslyverydifferentfromflagellaandfimbriae(113).Allthesesurfacestructuresmayhaveintracellularorigins;78[VOL.25 ANATOMYOFBACTERIALSURFACEFigure2.FimbriaearrangedaroundthecellsurfaceofShigellaflexneri(DuguidandGillies(28)).X45,000.flagellaapparentlypassthroughthewallandareresponsiblefortheadhesivepropertiesofmembraneandterminateinasphericalorganellecertainbacteria(28),thefunctionstheymaylocatedinthebacterialprotoplasm,asillus-conferonthecellhavenotbeenclearlyestab-tratedinfigures1and2.Althoughthefimbriaelished.791961J M.R.J.SALTONIII.BACTERIALCAPSULESCapsuleswereamongthefirstsurfacestruc-turestoberemovedfrombacterialcellsandchemicallycharacterized.Theclassicalinvestiga-tionsofAveryandHeidelberger(seeHeidelberger(43))onthechemistryandimmunochemistryofthepneumococcalcapsularpolysaccharideswerethefirstextensivestudiesgivingussomeconceptofthevarietyofcompoundsformingthesurfacelayersofcertainbacterialcells.Untilcomparativelyrecenttimes,thebacterialcapsulehasgenerallybeenregardedasahomoge-neousaccumulationofviscousmaterialaroundthecell.However,Tomesik's(110)studiescom-biningphase-contrastmicroscopyandantigen-antibodyreactionshaveshownthatthecapsulesofBacillusanthracisandcertainstrainsofBacillusmegateriumarebynomeansofsimplephysicalstructure.Indeed,theseinvestigationshaverevealedthepresenceoflocalizedpatchesofacapsularpolysaccharideinagelofcapsularglutamylpeptide.TheelectronmicroscopicstudybyLabawandMosley(59)hasestablishedaverycomplexphysicalstateforthecapsuleoftheLisbonnestrainofEscherichiacoli.Macro-molecularcomponentswereembeddedinastructurelessgelformingthecapsuleofthisorganism.StainedpreparationsusuallygivetheimpressionthatthecapsularsurfaceissmoothandcontinuousbutafurtherphysicalcomplexityIabwasbroughttolightwhenIvanovicsandHor-vath(55)detectedfairlyregularindentationsalongthesurfaceofthecapsuleofaBacillusmegateriumstrain.Thereisprobablylittledoubtthatthecapsulesofmanybacteriaarephysicallyhomogeneousstructuresofonetypeofpolymericsubstance,butwithrefinedmethodsofstudyingtheanatomyofthebacterialsurface,someofthemorecomplicatedstructures,illustrateddia-gramaticallyinfigure3,havenowbecomewellestablished.Therelationshipofthecapsulestotherigidcellwallhasbeenofgreatinterestandtheisola-tionofspecificcapsule-degradingenzymes(6,23)hasbeenoftheutmostvalueinstudyingthesur-faceanatomyofbacteria.Thereisnowavarietyofenzymesorenzymesystemsavailablefortheselectiveremovalofbacterialcapsules,leavingtheviabilityofthedecapsulatedcellsunim-pairedaswellastheabilitytosynthesizethecapsularmaterial.Encapsulatedpneumococci(6,23),klebsiellae(1),streptococci(63,67),andBacillusspp.possessingy-glutamylpeptides(112)havebeenenzymicallydecapsulated,thusestablishingtheanatomicalrelationshipsofthecapsulesandwallsoftheseorganisms.A.ChemicalCompositionChemically,bacterialcapsulesarepolymericsubstancesofeitherpolysaccharideorpolypep-cdFigure3.Adiagrammaticrepresentationofthetypesofcapsularstructure(takenfromreference88):(a)capsuleformingcontinuouslayeraroundcell;(b)capsularlayerwithbandedfibrils(59);(c)complexcapsulewithlocalizedpatchesofpolysaccharideandpolypeptide(10);(d)discontinuitiesincapsularsurface(55).80[VOL.25 ANATOMYOFBACTERIALSURFACETABLE1ChemicalnatureofbacterialcapsularsubstancesOrganismGram-positive:BacillusanthracisBacillusmegateriumBacilluscirculansPneumococciStreptococciGroupsAandCRumenspeciesGram-negative:AcetobactercapsulatumHaemophilusinfluenzaeAerobacter-KlebsiellagroupEscherichiacoliClassofSubstanceandProductsofAcidHydrolysisPolypeptide:-y-D-glutamylpeptidePolypeptide:-y-glutamylpeptidePolysaccharides:aminosugars;sugarsPolysaccharide:glucose,mannose,uronicacidPolysaccharides:sugars,aminosugars,uronicacids,ribitolphosphatePolysaccharide:hyaluronicacid-glucosamine,glucuronicacidPolysaccharide:galactose,rhamnose,uronicacidPolysaccharide:dextrin-glucosePolyribophosphatePolysaccharides:complexpolyuronides-glucose,fucose,glucuronicacidPolysaccharide:fucose,galactose,hexuronicacidDatasummarizedfromreferences88(givingoriginalreferences),42,78,127.tidenature.SofarasIamaware,heteropolymerscontainingpolysaccharideandpeptideresiduescovalentlylinked(asinbacterialwalls)havenotbeenencounteredascapsularsubstances.Ofallthebacterialcapsularpolysaccharidesstudied,thoseofthepneumococcihavebeeninvestigatedingreatestdetailandthechemicalstructureofanumberofdifferenttypeshasbeendetermined(43).Bothaminosugarsanduronicacidsarewidelydistributedincapsularpolysaccharidesfrombothgram-positiveandgram-negativebacteria.Thecapsularpolysaccharidesofgram-negativebacteriamustnotofcoursebeconfusedwiththepolysaccharidemoietiesoftheprotein-lipid-polysaccharidecomplexesconstitutingthemacromolecularcomponentsofthewallorenvelope.Thevarietyofmaterialsformingbac-terialcapsules(brieflysummarizedbySalton(88))isillustratedintable1andincludeshyal-uronicacid,polyuronides,variouspolysac-charides,andthey-glutamylpeptides.Thelistofcapsularsubstancespresentedintable1isbynomeanscomprehensive.Itdoesnotinclude,forexample,otherinterestingsurfacepolymerssuchastheaminouronicacidformingtheViantigen(16),theMproteinsofstreptococci(60),andothercomponentsthatarenotdemonstrablebythemethodsusuallyemployedfordetectingcapsules(26).Ingeneral,thereislittlechemicalrelationshipbetweencapsularsubstancesandthecell-wallstructures,therebeingdistinctivecompoundsinthewallenablingustodifferentiateonefromtheother.However,Naturehaspreventedusfromputtingeverythingintotightlittlecompartmentsandtherearenowtwoinstancesinwhichcapsularsubstanceshavebeenfoundtocontaincom-poundsthatwehavecometoregardasexclu-sivelywallsubstances.Anextremelyinterestingexampleofthissituationhasarisenfromthediscoveryofribitolphosphateinthespecificpolysaccharideoftype6pneumococcusbyRebersandHeidelberger(78).Uptothetimeofthisreport,ribitolphosphatecompoundsinpoly-mericformhadbeenfoundonlyintheteichoicacidsofthebacterialwall,discoveredbyBaddileyandhiscolleagues(4,9).Thesecondexampleofamaterialofcapsularorigincontainingawallsubstance,probablythecell-wallaminosugar,muramicacid,isthepolysaccharidederivedfromBacillusmegateriumbyGuex-HolzerandTomcsik(42).Thismaterialappearedtobeimmunologi-callyidenticaltothecell-wallsubstanceanduponisolationthecapsularpolysaccharidewasfoundtocontainglucosamine,galactosamine,andanunknownaminosugarpresumedtobemuramicacid.Whetherthismaterialshouldberegardedasatruecapsularpolysaccharide,orwhetheritrepresentsalocalaccumulationofwallmaterialbeing"over-produced"bythedividingcell,couldonlybedecidedbyfurtherinvestigation.Atleastthesetwocasesillustrate8119611 M.R.J.SALTON[o400450500WAVELENGTH(MP)550600Figure4.AbsorptionspectraoftheproductsofDische(22)reactionsonAerobacter(Klebsiella)aerogenesstrainA3untreatedwalls(0-0);wallsafterextractionofcapsularpolysaccharide(AA);andthehot-waterextractablepolysaccharide(E).E,cmfor0.45mgeachfraction(89).thepointthattheremaybeagreaterchemicaloverlapbetweencapsularsubstancesandwallsthanwehadhithertosuspected.AlthoughboththecellwallsandthecapsularpolypeptidesfromBacillusanthracisandBacillusmegateriumcon-taintheD-isomerofglutamicacid(52,54,84),thecapsularpeptideisasimplepolymer,whereasinthewalltheD-glutamicacidischemicallylinkedinamorecomplexmucopeptide.Theretentionofcapsularandsurfacesub-stancesonthecellwallduringitsisolationpre-sentsaprobleminestablishingthechemicalanatomyofthesurfacestructures.ThustheMproteinwasretainedonisolationofthewallsofagroupAstreptococcusanditcouldberemovedenzymaticallyfromthewallsbydigestionwithtrypsin(80).FurtherstudieswithacapsulatedstrainofAerobacter(Klebsiella)aerogeneshavealsoshownthatduringwallisolationsomeofthepolysaccharideisretainedand,onhydrolysisofthe"wall"fractions,monosaccharidescharacter-isticofthecapsularsubstancearealsopresent.Fortunatelywiththisstrainitispossibletodis-tinguishbetweenconstituentsofthecapsule(127)andthewallpolysaccharidecomponents,forDudmanandWilkinson(25)hadshownthatthepolysaccharidecouldbeextractedfromintactcellswithhotwater.Whenthe"wall"fractionswereextractedinthisway,thefucoseanduronicacidofthecapsularpolysaccharidewerefoundinthewater-solublefractions,leavingtheinsolublewallfractiondevoidofthesesugars(89).TheDische(22)reactionformethylpentosecanbeusedtofollowthedistributionofthesesugarsin"wall"andcapsularpolysaccharidefractionsasillustratedinfigure4.Themonosac-charideconstituentsdetectedin"wall"fractionsofencapsulated,slime-producing,andnonen-1.00.90.80.70.60.5Uw0.40.3a0.20.1082[VOL.257wwAll-..AAA4"A-AAAAm=- ANATOMYOFBACTERIALSURFACETABLE2Monosaccharideconstituentsof"walls"ofen-capsulated,slime-producing,andnonencapsulatedstrainsofAerobacter(Klebsiella)aerogenesStrainPreparationMonosaccharidesA3capsulatedCellwallsGalactose,glu-cose,fucose,uronicacidWallextractedGalactose,glu-withhotcosewaterExtractedGalactose,glu-capsularcose,fucose,polysac-uronicacidcharideA3(S)slime-WallsGalactoseproducingA3(0)non-WallsGalactoseslime,non-capsularReference89.capsulatedstrainsofAerobacter(Klebsiella)aerogenesaresummarizedintable2.Theseresultsemphasizetheneedforspecificremovalofcapsularsubstanceswhenadifferentiationofwallandcapsularpolysaccharidesisbeingsought.IV.THECELLWVALLThemajorstructuralcomponentofthebac-terialcellistherigidwall,whichmayaccountforabout10to40percentoftheweightofthecell(88).TheexcellentstudiesofShockman,Kolb,andToennies(100)havedemonstratedhowthewallcontributiontotheweightofthecelldependsonthegrowthphase,risingfrom27percentintheexponentialphaseto35percentinthestationaryphaseoftheorganism,Strepto-coccusfaecalis.Furthermore,thenutritionalstatusoftheorganismcanalsoinfluencetheamountofcell-wallsubstanceformed,asshownintheaminoaciddepletionstudiesbyShockman(99)-Theisolationandcharacterizationofbacterialcellwallshasinrecentyearspresenteduswithsomefascinatingdetailsofthecomparativeanatomyandchemistryofgram-positiveandgram-negativebacteria.Fromthevarietyofwallsexaminedintheelectronmicroscope,itisnowapparentthat,ingeneral,thewallsofgram-negativebacteriaarephysicallymorecomplexthanthoseofgram-positiveorganisms(90).ThepresenceofsphericalmacromoleculesinacellwallwasfirstreportedbyHouwink(48).Sincethenanumberofspirillaandothergram-negativebacteriahavebeenshowntopossessfinestructureinthewalls(seeSalton(90)).Layersofhexag-onallypackedsphericalmacromoleculeshavebeenencounteredmostfrequently.AlthoughtheisolatedwallofEscherichiacolihasahomoge-neousappearanceonexaminationofshadowedpreparationsintheelectronmicroscope(48,93),itisevidentfromthebeautifulthinsections,preparedbyKellenbergerandRyter(57)andpresentedinfigure5,thatthewallismulti-layered.ThestudiesofWeidelandhiscolleaguesarenowbeginningtocorrelatethephysicalandchemicalcomplexityofthewallofthisorganism(125).Atypeoffinestructuredifferingfromthatcommonlyfoundinmanyofthegram-negativebacteriahasbeenobservedinthecomplexwallor"envelope"oftheorganism,Lampropediahyalina.ThismacromolecularstructureobservedindependentlybyJ.A.ChapmanandM.R.J.Salton(unpublishedobservations)andR.G.E.Murray(unpublishedobservations)isillustratedinfigure6.A.ChemistryofCellWallsThefirstattempttodiscoverthechemicalcompositionofabacterialcellwallwasmadeaslongagoas1887byVincenzi(116).Apartfromfindingsubstantialamountsofnitrogenanddeducingthatthewallwasnotcomposedofcellulose,Vincenzi(116)wasunabletosuggestanythingofamoredefinitenature.However,duringthepasttenyearsagreatdealofinforma-tiononthechemicalcompositionofbacterialcellwallshasbecomeavailableandseveralout-standingfeatureshaveemerged.Thewallsofgram-negativebacteriawerefoundtobechemi-callymorecomplexthanthoseofgram-positiveorganisms(81).Wallsofgram-positivebacteriacontainedasmallvarietyofaminoacids,aminosugar,andsugarcomponents(81).Itwasthusapparentthatthewallsofgram-positivebacteriaweremadeupofanewstructuralclassofpoly-mersdifferingfromthestructuralpolysac-charidescommonlyencounteredinthewallsoffungiandhigherplants.Thestudyofthechemistryofbacterialcellwallshasattractedmanyinvestigatorsinthepastdecadeandonlythebriefestaccountcanbegivenofsomeoftheessentialfeatures.Several1961]83 4M.R.J.SALTON;..;r.;".:.*0........"_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~IFAf.Figure5.ThinsectionsofEscherichiacolistrainBinfectedwithT2phageshowingmultilayeredcellwallandaseparateelectron-denselayer,thecytoplasmicmembrane(KellenbergerandRyter(57)).X63,000.substancesnotgenerallyencounteredinthecellsofhigherorganismshavebeenfoundinbacterialcellsandshowntobelocalizedintherigidwallstructures.Thusmuramicacid(104),discoveredfirstinsporepeptidesbyStrangeandPowell(106)andlaterinwallsbyCumminsandHarris(19),StrangeandDark(105),andSalton(83),anda,e-diaminopimelicacid,isolatedandcharacterizedbyWork(128),aretwosubstancesconfinedtobacteriaandcloselyrelatedmicro-organismssuchasblue-greenalgae(90,130).Anotherfascinatingfeatureofcell-wallchemistryhasbeenthewidespreadoccurrenceoftheD-isomersoftheaminoacids,alanine,glutamicacid,andasparticacid(52,53,84,99,109),inthewallmucopeptides.IfwecanendowNaturewithpurpose,itseemseminentlysensibleofHertohavedesignedwallscontainingD-aminoacidsintheirpeptides,thusprovidingstructuresresistanttomanyofthecommonerproteolyticenzymesandpeptidases.Thewallsofmanygram-positivebacteriamaybemadeupentirelyofthemucocomplexsubstances,mucopeptidesandmucopolysac-charides,orboth.Themucopeptides(64)invari-ablycontaintheaminosugarsglucosamineandmuramicacid,andpeptidescomposedofavarietyof3,4,or5principalaminoacids.Inadditiontomucopeptides,somewallscontainpolysaccharideoroligosaccharideresidues(46,84[VOL.25 ANATOMYOFBACTERIALSURFACEFigure6.FinestructureinawalllayerobtainedfromdisintegratedcellsofLampropediahyalina(unpublishedobservationsbyJ.A.ChapmanandM.R.J.Salton).X110,000.68,79)covalentlylinkedtothemucopeptides.Anewclassofcell-wallpolymerdifferingfromthemucocomplexsubstanceswasdiscoveredseveralyearsagobyBaddileyandhiscolleagues(4,5,9).Followingthecharacterizationoftwonucleotides,cytidinediphosphoglycerolandcvtidinediphosphoribitol(8),asearchforabio-syntheticfunctionforthesenucleotidesledtothedetectionofribitolandglycerol-phosphatepolymersinbacteriaandultimatelytothelocalizationoftheteichoicacids(fromGreekteichos=wall)intheisolatedcellwalls.ItwillberecalledthatinanearlierstudyMitchellandMoyle(72)hadfoundpolyolphosphatesintheir"envelope"fractionsofStaphylococcusaureus.Ingeneralthetwotypesofteichoicacid(glycerol-andribitol-teichoicacid)donotoccurtogether(5)andtheyseemtobeabsentfromanumberofgram-positivebacteria.Oneinterestingfeatureoftheteichoicacidsisthepresenceofester-linkedalanineinbothtypes.Therelationshipoftheglycerol-teichoicacidtothepolyglycerophos-phatecompoundfoundinanumberofgram-positivebacteriabyMcCarty(69)hasnotbeenestablished.Thelattercouldconceivablyarisefromtheglycerol-teichoicacidifthelabileester-linkedalaninewaslostduringisolationandpurification.Studiesonthemolecularstructureofwallmucopeptideshaveadvancedrapidlyinthepastfewyears.Muchinformationhasbeengainedfromseveralsources,includingtheelucidation8519611 M.R.J.SALTONBACKBONESTRUCTURE(1-6)(1-4)(16)(14)(16)(1-4)(16)AG-AMAAGAMAAG-AMA.AGAMA.PeptidePeptideLsozymeSensitivebondsFigure7.TypeofmolecularstructureproposedforthewallofMicrococcuslysodeikticus(91),showingthearrangementofpeptideside-chainsonanacetylaminosugarbackbonepossessingalternating1-44,1-46bondsbetweenN-acetyl-muramicacid(AMA)andN-acetylglucosamine(AG).ofthestructureofthenucleotidesaccumulaitnginthepresenceofpenicillinandotherantibiotics(77,107)andinvestigationsoftheproductsofenzymatichydrolysisofisolatedcellwalls(83).Onetypeofstructureemergingfromtheearlyworkontheproductsoflysozymedigestionofwalls(83)wassuggestedbySalton(85)andfurtherexpandedfromtheknowledgeofthenucleotidestructurebyBrumfitt,Wardlaw,andPark(14).Theisolationofdi-andtetrasac-charidesandpeptide-aminosugarcomplexes(mucopeptides)inlysozymedigestsofwalls(35)andtheirchemicalcharacterization(34,91)enableustosuggest(92)thetypeofstructureforthewallofMicrococcuslysodeikticusillustratedinfigure7.Thecellwallsofgram-negativebacteriaaremuchmorecomplex.Inadditiontocontainingmucopeptidesincommonwithgram-positivebacteria(87,124)theyalsopossessmajorpro-tein,lipid,andpolysaccharideconstituents.Themucocomplexormucopeptidecomponentsofthewallsofgram-negativebacteriamayaccountforonly10to20percentoftheweightofthewall,butitisapparentthatitisthisclassofchemicalconstituentwhichisresponsibleforthestructuralrigidityofthewall(87,88,90,124,125).Itseemslikelythattheprotein,lipid,andpolysaccharidecomponentsofthewallarepresentasamacro-molecularcomplex,withthemucopeptideform-inglinksinarigidlayer(125)orareinforcingnetworkthroughouttheentirewall(90).Thevariousantigenicsubstancesisolatedasthe0,orsomaticsmooth-phase,antigensareprobablyderivedfromthemacromolecularcomplexesofthecellwalls(89).Itisnotknownatpresentwhetherthewallismadeupofavarietyofchemicallyandimmunologicallydifferentmacro-molecularsubunits.TABLE3ChemicalcompositionofbacterialcellwallsGram-posi-tivebacte-riaGram-nega-tivebacte-riaPrincipalClassesofConstituentsandProductsofAcidHydrolysis1.Mucopeptides-glucosamine;muramicacid;3,4,or5aminoacids.2.Mucopolysaccharides-aminosugars,monosaccharides3.Teichoicacids-ribitol,phos-phate,glucoseorglucosamine,alanine;glycerol,phosphate,alanineWallsmayhavecompositionswiththefollowingcombina-tions:1,1+2,1+3,1+2+3.Mucopeptidesasabove.(Teichoicacidsnotasyetisolatedfromthisgroup.)ProteinIProbablyLipid(presentascom-PolysaccharidesplexesofallthreeclassesReferences88,90.Someoftheprincipalfeaturesofthechemicalcompositionofbacterialwallsaresummarizedintable3.Moreextensiveaccountsofcell-wallchemistryareavailableinearlierreviewsbyCummins(18)andWVork(129)andinmorerecentcontributionsbySalton(88,90).V.PROTOPLASTMEMBRANESOneofthemostimportantadvancescontribut-ingtothefurtherstudyoftheanatomyofthesurfacestructuresofthebacterialcellwasmadewhenWeibull(118)isolatedandcharacterizedtheprotoplastsofBacillusmegateriumin1953.Thisenabledadirectexaminationofthefunc-tionalandchemicalpropertiesofthebacterialmembranetobemade.Thattheisolatedproto-plastpresentedasurfacedifferentfromthatoftheoriginalintactcellwasdemonstratedinanumberofways.Itcouldnotbeinfectedwithbacteriophages(118),whichrequireaspecificreceptorinthecellwall(94).Theantigensonthesurfaceoftheprotoplastsdifferedfromthoseoftheisolatedwallsandintactcells(111,114,115).Unlikewallsandintactcells,theprotoplastsofBacillusmegateriumandMicrococcuslysodeikticusandtheirmembranesareextremelysusceptibletodisaggregationwithsodiumdodecylsulfate86[VOL.25 ANATOMYOFBACTERIALSURFACEandotheranionicdetergents(36,38,86).Thesevariouspropertiesbasedonthecharacteristicsoftheprotoplastmembranecanbeusedascriteriaindefiningprotoplasts,atermreservedfortheorganizedprotoplasmicelementsofbacterialormicrobialcellsdeprivedcompletelyofthecell-wallstructure(12).Mostofthetrueprotoplastshavebeenobtainedbyenzymaticdegradationofthewall(12,20,32,40,70,118).Withorgan-ismssuchasBacillusmegaterium,Micrococcuslysodeikticus,andSarcinalutea,walldegradationwithlysozymemaybecompleteandleavenoneofthecharacteristicwallcompoundsintheprotoplastsortheprotoplastmembranes(12,70).Thereare,however,severalinstancesinwhichprotoplastshavebeenobtainedwithoutcom-pletedigestionofthecellwall.AnautolyticenzymefromStaphylococcusaureuscutsthewallofthatorganismintotwohemispheres,thusreleasingtheintactprotoplastwhentheen-zymaticactionisallowedtotakeplaceinasuitablestabilizingmedium(75).Similarly,partialbreakdownofthewallofNeurosporacrassapermitstheextrusionofanintactproto-plast(7).Unfortunately,notallorganismsareamenabletosuchelegantenzymaticmanipulationsde-signedforthestepwise"peelingoff"ofthesur-facelayersofthecell.Thewallsofmanygram-positivebacteriaareonlypartiallydegradedbylysozyme(95)and,consequently,attemptstoisolateandcharacterizetheprotoplastmem-branesoftheseorganismswillhavetoawaitthedevelopmentofmorespecificenzymeprepara-tions.Theincompleteremovalofwallcom-ponentsisespeciallyconspicuouswiththeforma-tionofsphericalcells("protoplasts,"spheroplasts(12,70))ofgram-negativebacteriafollowingtreatmentwithlysozymeandethylenediamine-tetraaceticacid(EDTA),orthegrowthoftheseFigure8.Someanatomicalconsequencesoftheactionofpencicillinonbacteria.ThinsectionsofStaphylococcusaureusexposedtopenicillin.Grossdistortionatthepointsofcrosswallfor-mationclearlyvisible(Murray,Francombe,andMayall(76)).X46,000.1961187 M.R.J.SALTONFigure9.Someanatomicalconsequencesoftheactionofpenicillinonbacteria.Vibriometschnikovii"protoplasts"preparedbygrowthinthepresenceofpenicillin.Themajorityoftheprotoplastssoformedhaveanouterweakwallasintheleft-hand"protoplast";theright-handonehashadthewalldetachedduringpreparationforelectronmicroscopy(Salton(90)).X16,500.organismsinthepresenceofpenicillin(61,70,96).Aspointedoutpreviously,themucopeptidecomponentofthewallofgram-negativeorgan-ismscanberegardedaseitherareinforcingnetworkor,assuggestedbyWeidel,Frank,andMartin(125),aspartofanorganized,rigidlayer.Atleastthestructuralconsequencesoftheactionofpenicillininitsinterferencewiththeformationofmucopeptidecanbeclearlyseenfromthe"lesions"apparentinthethinsectionsofStaphylococcusaureusshowninfigure8,takenfromthestudiesofMurray,Francombe,andMayall(76).Suchanorganismhas,sotospeak,nosecondlineofdefenseinitswall,foroncethemechanicalintegrityofthewallisbreachedtheprotoplastmembranewillbeunabletowithstandthehighosmoticpressureandlysiswillultimatelyensue.Bywayofcontrast,thewallsofthegram-negativebacteriawiththeirprotein-lipid-polysaccharidecomplexeshaveanadditionalchanceofmaintainingtheirintegrity.Althoughthewallisconsiderablyweakenedbygrowthinthepresenceofpenicillin,thetypical"poached-egg"appearanceofthesphericalformsofVibriometschnikoviistillshowsanouterwallsurround-ingtheprotoplast(figure9).Unliketheproto-plastsofgram-positivebacteria,thesesphericalcellsorspheroplastsareagglutinatedbyintactcellandcell-wallantisera,thusindicatingaverysimilar,ifnotidentical,immunologicalsurface(47,96,98).Thestructuralanalysisofgram-negativebacteriahasthusbeenhamperedbytheabsenceofsuitablemethodsforisolatingprotoplastsanalogoustothoseofgram-positivebacteriaandasaconsequencetheevidencefortheexistenceofaseparate,functionalprotoplasmicmembranestructureisstilllargelycircumstantial.However,88[VOL.25 ANATOMYOFBACTERIALSURFACEtheappearanceofthinsectionsofgram-negativebacteriastronglysupportstheconclusionthattheytoopossessawelldefinedprotoplastmem-brane(seefigure5).Thebehaviorofthe"envelope"or"hull"ofcertaingram-negativeorganismshassuggestedtoseveralinvestigatorsthatseparatewallandmembranestructuresmaynotexistinthem(65,66).Itislikelythatthisquestioncouldbere-solvedwithanenzymesystemcapableofdegrad-ingthewallsufficientlytoallowtheisolationofaprotoplastifsuchexists.AttemptstoisolatesuchenzymeshavebeendisappointingandaspointedoutbySalton(82)theproductionbyasingleorganismofalltheenzymesrequiredtobreakdownthewallmaybeaninfrequenteventinNature.Itisofcourseapparenttoeventheele-mentarystudentofmicrobiologythattheremustbeorganismsproducingenzymescapableofbreakingdowneverything(usedonlyinthesenseofallmacromolecularcomponentsandtheirbuildingblocks),otherwisewewouldbekneedeeporevenfurtherimmersedinwallsofgram-negativebacteria!Whensuchenzymesbecomeavailableitmaybepossibletodecidewhetherthereisastructureanalogoustothemembraneofthegram-positivebacteria,orwhetherthe"envelope"ofthegram-negativeorganismsisanintegratedstructurepossessingboth"wall"andtransportingfunctionsofanosmoticmembrane.Thesuccessofthisapproachwouldbedependentontherebeingenoughdifferenceinthechemicalconstitutionofwallandmembranetopermittheselectionofspecificenzymes.Itisquiteconceiv-able,however,thatwallsandmembranesofgram-negativebacteriamaybesufficientlysimilartomaketheselectiveremovalofthewallverydifficultorevenimpossible.A.ChemicalCompositionofMembranesThattheprotoplastmembranewouldbechemicallymorecomplexthanbacterialwallswassuspectedfromWeibull's(118,119)demon-strationofthepresenceofthecytochromesystemintheisolatedmembranesofBacillusmegateriumandthedetectionofanumberofenzymesoftheelectrontransportsysteminthesestructuresbyStorckandWachsman(103).TwogroupsofinvestigatorshaveisolatedandcharacterizedchemicallytheprotoplastmembranesofBacillusmegaterium(122)andMicrococcuslysodeikticus(39).BothbacterialmembranesaremadeupTABLE4ComparisonofcompositionofcellwallandprotoplastmembraneofBacillusmegaterium%DryWeightConstituentWallMembraneNitrogen..................7.4-7.810.3-10.9Phosphorus.............3...3.43.5Lipid....................4.2-5.915.9-20.9Hexose....................0.3-0.91.8-9.8Aminosugar................20-23Diaminopimelicacid.7-9.Reference122.TABLE5ComparisonofcompositionofcellwallandprotoplastmembraneofMicrococcuslysodeikticus%DryWeightConstituentWallMembraneNitrogen....................7.68.4Phosphorus..................0.221.16Lipid......................028.0Mannose....................018.9Glucose.....................3.5-5.80Aminosugar.................16-222.7Reference39.largelyofproteinandlipid.Acomparisonofwallandmembranecompositionofeachspeciesispresentedintables4and5.Thelipidofthemembranesofthesetwoorganismsappearstobemainlyphosphatidicacid;accordingly,GilbyandFew(38)havesuggestedthatcationicdeter-gentsmayactonthislipidcomponentofthepro-toplastmembrane.Themembranefractionshavefrequentlybeenfoundtoformacharacteristicyellowlayeroncentrifugationoflysedproto-plasts(39,70,75,118).Thispigmentationcanatleastinpartbeaccountedforbythepresenceofcarotenoids(37,39).Nucleicacids(bothribo-nucleic(RNA)anddeoxyribonucleic(DNA))havebeendetectedinisolatedmembranes(114,122).WeibullandBergstr6m(122)foundthattheRNAcontentsofbatchesofmembranesofBacillusmegateriumvariedfromabout0.5to2percentbutsubstantiallyhighervalueshavebeenobservedbyVennesandGerhardt(114).It1961]89 M.R.J.SALTONispossiblethatthenucleicacidmaterialpresentinthemembranefractionmayrepresentcon-taminatingmatterfromthebacterialprotoplasm(122).VI.LOCALIZATIONOFENZYMESINBACTERIALCELLSANDASUMMARYOFTHECOMPARATIVECHEMICALANDBIOCHEMICALANATOMYOFGRAM-POSITIVEANDGRAM-NEGATIVEBAC-TERIAWiththemethodsavailableforthereleaseofprotoplastsofgram-positivebacteriaandtheisolationoftheprotoplastmembranesithasbeenpossibletocometosomeconclusionsaboutthelocalizationofcertainenzymesinthemajorsurfacestructuresofthebacterialcell.Thebio-syntheticcapabilitiesofthebacterialprotoplast(70,120)aresosimilartothoseofintactcellsthatitappearsunlikelythatthecellwallcon-tributesmuchmorethanmechanicalstabilitytothebacterialcell.Thusthecompletelossofthewallduringprotoplastformationdoesnotseriouslyimpairthefunctioningoftheosmoticbarrier,thebiosynthesisofcomplexmoleculessuchasproteinsandnucleicacids,orthesyn-thesisandassemblyofbacteriophagesandspores(20,31,120).ThedistributionofenzymesinprotoplastandsolubleandparticulatefractionsofBacillusmegateriumhasbeenstudiedbyseveralinvesti-gatorsandthereisverygoodagreementbetweentheresultsofStorckandWachsman(103)andthoseofWeibull,Beckman,andBergstrom(123)forseveralstrainsofthisorganism.SomeoftheresultsforthelocalizationofenzymesinTABLE6RelativeamountsofenzymesinmembraneandsolubleprotoplasmicfractionsofBacillusmegateriumEnzyme~~MembraneSolubleEnzymeFractionProtoplasmSuccinicdehydrogenase......145.04.5Malicdehydrogenase.........32.615.7Lacticdehydrogenase........41.257.2Isocitricdehydrogenase3.5112.0Fumarase....................32.2101.8DPNHoxidase..............261.01.0Catalase.....................1.3100.0Hexokinase..................5.587.5Acidphosphatase............3.099.6Datafromreference123.TABLE7EnzymedistributioninfractionsfromastrainofPseudomonasdisintegratedintheMickleapparatusCrudeProto-EnzymeWallplasmicFractionFractionSuccinicdehydrogenase........+:i1Malicdehydrogenase...........+4Fumaratedehydrogenase+Alaninedehydrogenase.-.+DPNHoxidase................+TraceUnpublisheddata,A.D.Brown,S.Jeffery,andM.R.J.Salton.thestudyofWeibulletal.(123)arepresentedintable6.InvestigationswithBacillusmegaterium(123)andStaphylococcusaureusmembranes(74)haveconfirmedthepresenceofcytochromes,thereduceddiphosphopyridinenucleotide(DPNH)oxidase,thesuccinicdehydrogenase,andthemalicdehydrogenasesystemsinthesestructures.MitchellandMoyle(74)foundacidphosphatasemainlyinthemembraneofStaphylococcusaureus,whereasthisenzymeandhexokinasewerelargelyinthe"soluble"protoplasmicfractionofBacillusmegaterium(123)asshownintable6.Thereisnoinformationaboutthedetectionofenzymesinwallfractionsofgram-positivebacteria.Thechemicalcompositionofthesestructureswould,however,leadonetosuspectthatwhateverenzymeswerepresentcouldbederivedfromcon-taminationwithmembranefragmentsorad-sorbedprotoplasm.Thisofcoursedoesnotpre-cludethepossibilitythatenzymesarelocatedonthewalloftheintactbacterialcell.Owingtothedifficultyofisolatingmembranesofgram-negativebacteriaasseparatestructuralentities,thequestionwhethercertainenzymesarelocalizedinthewallormembranewillhavetoawaitfurtherinvestigation.However,therehavebeenseveralstudiesofthedistributionofenzymesinsoluble,particulate,and"envelope"or"hull"fractionsofgram-negativebacteria(2,17,50,51,62).Anumberoftheenzymesoftheelectrontransportsystemhavebeenfoundinthe"envelope"fractions(17,51).Hunt,Rodgers,andHughes(51)isolateda"cellwall-membrane"fractionfrommechanicallydis-integratedcellsofastrainofPseudomonasfluorescens.Fromtheirexcellentstudiestheyconcludedthatthenicotinicacidhydroxylase90[VOL.25 ANATOMYOFBACTERIALSURFACEandsuccinicaciddehydrogenasesystemswerelocatedinthiscomplexstructure.Furthershak-ingofthese"wall-membrane"fractionswithglassbeadsdidnotreleasemuchoftheenzymeactivities.However,lysozymeandEDTAtreat-mentat25Cfor8to10minreleasedthetotalactivityintothesupernatantfraction.A.D.Brown,S.Jeffery,andM.R.J.Salton(unpub-lishedobservations)studiedthedistributionofenzymesincrude"wall,"smallparticle,andprotoplasmicfractionsofaPseudomonassp.disintegratedinbufferat0CintheMickleapparatus.Thequalitativeresultspresentedintable7againconfirmthepresenceofsuccinicaciddehydrogenase,malicdehydrogenase,andDPNHoxidaseinthecrudewallor"envelope"fractions.Owingtothegreaterdifficultyinob-tainingcleanwallfractionsofgram-negativebacteria,furtherinvestigationswillbeneededbeforeitispossibletoarriveatfirmconclusionsonthedistributionofenzymesinthewallandmembranestructuresorparticlesderivedfromboth.Atthemoment,allwecandecideaboutthegram-negativeorganismsiswhetheranenzymeispresentinthe"envelope"(cellwall-membrane)fractionorintheparticulateorsolublefractions.GRAM-POSITIVECAPSULEPOLY-SACCHARIDEPOLY-PEPTIDEWALLMUCO-PEPTIDEMUCOPOLY-SACCHARIDETEICHOICACIDSENZYMESIMEMBRANEPROTEINLIPIDPOLY-SACCHARIDEENZYMESCYTOCHROMESDEHYDROGEN-ASESDPNH-OXIDASEAlthoughtherearestillmanyseriousgapsinourknowledgewecansummarizeanumberoftheessentialfeaturesofthecomparativeanatomyofthesurfacestructuresofgram-positiveandgram-negativebacteria.Someofthechemicalandbiochemicalpropertiesarepresentedinfigure10.VII.THEGRAMSTAINANDTHEBACTERIALSURFACEWithourpresentincreasedknowledgeofthenatureofthebacterialsurface,isitpossibletoconcludeanythingmoredefinitiveaboutthemechanismoftheGramstainreaction?IbelievewearemuchclosertoanunderstandingofthisstainprocedurewhichdividesthebacterialworldintotwobroadgroupsseparablenotmerelyontheirresponsetotheGramstain,butalsoonthebasisofbiochemicalandchemicalproperties(10).ItshouldofcoursebepointedoutthatitisnowalmostimpossibletoproposeanynewtheorytoexplaintheGramstainreaction,asmostofthepossibilitieshavebeencoveredatsometimeorotherduringthelonghistoryofthisstainingprocedure.Moreover,everymajorclassofthemacromolecularcomponentsofthebacterialcellhasbeenimplicatedinthemechanismoftheGRAM-NEGATIVECAPSULFPOLY-SACCHARIDEWALLPROTEINLIPIDPOLY-SACCHARIDEMUCO-PEPTIDEMEMBRANECOMPOSITIONENZYMESENZYMESa??jENVELOPEENZYMESCYTOCHROMESDEHYDROGENASESDPNHOXIDASEFigure10.Summaryofthechemicalandbiochemicalanatomyofthesurfacestructureofgram-positiveandgram-negativebacteria.911961] M.R.J.SALTONTABLE8TheoriesandcellularsubstancesinvolvedintheGramstainreactionNucleoproteinsDeussen(21)NucleicacidsDubosandMacLeod(24)Henry,Stacey,andTeece(44,45)LipidsEisenberg(30)SpeciallipidsSchumacher(97)Lipo-proteinStearnandStearn(102)CarbohydrateandWebb(117)nucleicacidGlycerophosphateSchumacher(97)complexPolyglycerophos-MitchellandMoyle(71)phatePermeabilityBurkeandBarnes(15)KaplanandKaplan(56)WensinckandBoev6(126)Bartholomew,Cromwell,andFinkelstein(11)stainprocedure.Table8givesabrief,selectedsummaryoftheprincipaltheoriesandsub-stancesallegedtobeinvolved(10,11,15,21,24,30,44,45,56,71,73,97,101,102,117).SoofteninthestudiesofthemechanismoftheGramstain,attemptshavebeenmadetoisolatespecificsubstancesthatmayberesponsiblefortheretentionofthecrystalviolet(CV)-iodine(I)complex.ThisapproachledHenry,Stacey,andTeece(45)to"restore"theGramstainwithextractedMg-ribonucleateandtoconcludethattheRNAofgram-positivebacteria,coupledtobasicproteins,wasresponsibleforthestainreac-tion.MitchellandMoyle(71)couldfindnocorrelationbetweentheGramstainandnucleicacidcontentsbuttheybelievedtheGramreac-tionwasrelatedtothepresenceofpolyolphos-phatesinthebacterialenvelope(71,73).Hereagainaconvincingcorrelationhasbrokendown,aswenowknowthatvariousstronglygram-positivebacteriaaredevoidoftheteichoicacidsinthewall.Thatmanysubstanceslikelytooccurinbacterialcellscanstainasgram-positivematerialhasbeenshownbyShugarandBaranow-ska(101).Lipids,polysaccharides,RNA,andcertainproteinscanallretaintheCV-Icomplextoagreaterorlesserextent(101).ItthereforeTABLE9Cell-wallcompositionandGramstainreactionGramMajorChemicalOrganismReac-ComponentsoftionCellWallsSaccharomyces+Polysaceharide,cerevisiaeprotein,lipidCandidaspp.+Polysaccharide,proteinStaphylococcus+Mucopeptide,aureusteichoicacidsBacillussubtilis+Mueopeptide,teichoicacidsStreptococcusfaecalis+Mucopeptide,mucopolysac-charide,teichoicacidsMicrococcuslyso-+MucopeptidedeikticusEscherichiacoli-Protein,polysac-charide,lipid,mucopeptideSalmonellagal--AsforE.colilinarumProteusvulgaris-AsforE.coliSpirillumserpens-AsforE.coliseemedunlikelythattheGramstaincouldbecorrelatedwiththepresenceofanyonespecificsubstanceinthecellsofthosebacteriashowingapositivereaction.TowhatextentthechemicalconstituentsofthebacterialwallarecorrelatedwiththeGramstaincanbejudgedfromthedatapresentedintable9.Themainfeatureswhichemergefromcomparativestudiesofthechemistryofmicrobialwallsarethepresenceofmucopeptide,mucopoly-saccharide,andpolysaccharidecomplexesinthewallsofallgram-positivebacteriaandtherelativelyhigh(upto20percent)lipidcontentsinthewallsofgram-negativeorganisms.Itthereforeseemedconceivabletothewriterthatontheonehandthehighlipidcontentofthewallsofgram-negativebacteriamightbeafactorcontributingtotheirnegativity,andthatontheotherhandthedehydrationofthewallmuco-complexesduringthedecolorizingstepoftheGramstainmightreducethe"poresize"inthewallandrendertheCV-Icomplexrelativelyinaccessibletothesolvent.Bothofthesepossi-bilitieswouldbeamenabletoexperimentationandanyresultsshouldgiveanindicationofthe92[VOL.25 ANATOMYOFBACTERIALSURFACEimportanceof"permeability"factorsintheGramstain.Theextractabilityoflipidsfromthewallswith95percentethanol,theconcentrationemployedinthedifferentiationstepinHucker'smodifica-tionofthestain(10),wastested.From40to50percentofthelipidcontentofisolatedwallsofEscherichiacoliorProteusvulgariscouldbeextractedundertheseconditions.ThedirectremovalofwalllipidfromtheseorganismscouldthuscontributetotheextractabilityoftheCV-Jcomplex.Ifthereisadifferentialresponseofthewallofthetwogroupstothepassageofsubstancesacrossitinethanol,thensomefurtherevidencecouldbegainedfrom"permeability"(notinaphysiologicalsenseofcourse)orleakagestudies.WithorganismsgrownonmediacontainingP32,ithasnowbeenpossibletoshowadifferentialpassageofintracellularmetabolitescontainingP32acrossthewallsor"envelopes"(cellwall-membrane)whencellswereplacedingradedcon-centrationsofethanol.Acomparisonoftypicalresultsobtainedwithtwogram-positiveandtwogram-negativebacteriaisaffordedbytheresultsshowninfigures11and12.Thisdifferentialeffectofethanolconcentrationontheleakageofintracellularsubstancesfromgram-positiveandgram-negativebacteriaisverysimilartotheextractabilityoftheCV-Icomplexreportedinz7-1-.-Uw(nw-Jwc'JC,)CLj20001000theexcellentstudiesofWensinckandBoev6(126).Theleakagestudiesthussuggestthattheporesizeofthewallissufficientlyreducedduringdehydrationinhighconcentrationsofethanoltotrapalargefractionoftheintracellularconstitu-entswithinthecellsofgram-positivebacteria.Aseriesofgram-positiveandgram-negativebac-teriahavebeenstudiedinthismannerandtheethanol-inducedreleaseofP32(expressedasapercentageofthemaximalreleaseforeachorganism)issummarizedintable10.Exposureofgram-positivebacterialabeledwithP32toiodinesolutions(asusedintheGramstain)priortosuspensioninethanolreducedtheleakageevenfurtherintheethanolconcentrationsbe-tween80to100percent(v/v).Suchapretreat-mentwaswithouteffectonthegram-negativebacteria.Leakageexperimentswerethenper-formedonwashedcellsusingtheGramstainconditionsforbacterialsuspensionsdescribedbyWensinckandBoeve(126),i.e.,suspensionincrystalvioletsolution,washingwithwater,andtreatmentwithiodine.WhenthecellsweretakenupingradedethanolconcentrationsandthePnreleasewasdetermined,thedifferentialpatternofextractabilityforthegram-positiveStaphylococcusaureusandthegram-negativeEscherichiacoli(asshowninfigures11and12)persisted.Theseresultsareinaccordwith"per-meability"differencesbeingresponsibleforthe02550750/(V/V)100ETHANOLFigure11.Effectofethanolconcentrationonthereleaseofp32compoundsfromStreptococcusfaecalis(SF)andSaccharomycescerevisiae(SC)cellsuspensionsat20C.1961]93 M.R.J.SALTON[EC2000100002550751000/0(VlV)ETHANOLFigure12.EffectofethanolconcentrationonthereleaseofP32compoundsfromEscherichiacoli(EC)andProteusvulgaris(PV)cellsuspensionsat20C.Gramstain.TheevidencesuggestsnotonlythattheCV-Icomplexislargelyinextractablebutalsothatevensmallmolecularweightmetabolitescon-tainingP32arealsotrappedwithinthecellwhenthewallisdehydratedwith95percentethanol.Ithasofcoursebeenknownforsometimethatcellstreatedwithlysozymebecomegram-negative(Webb(117))andinmorerecentyearsisolatedprotoplastshavealsobeenfoundtogiveagram-negativereactionasshownbyGerhardt,Vennes,andBritt(33).TheseworkershavealsoshownthatcrushedprotoplastsfromBacillusmegateriumGram-stainedpriortowallremovalwithlysozyme,aswellascrushedwholecells,couldbedecolorized,thusreaffirmingthatstruc-turalintegrityoftheorganismisaprerequisiteforGrampositivity.Finally,themostconvincingevidencethatitisthewallofthegram-positiveorganismwhichisthebarriertoremovaloftheCV-Icomplexhasbeenobtainedwithsuspen-sionsofseverallysozyme-sensitivebacteriaGram-stainedbytheproceduredescribedbyWensinckandBoeve(126).Whenstainedcellsuspensionsofgram-positiveorganismsareincubatedwithlysozyme,thecellwallsaredigestedandtheresidualprotoplastretainstheCV-Icomplex,whichisthencompletelyaccessi-bleandisextractedquantitativelybyasingleTABLE10Releaseofp32frombacteriain100percentethanolGramReleasedReac-OrganismReleatiedttionMaximum-Pseudomonassp.96-Proteusvulgaris90-Escherichiacoli84-Salmonellagallinarum75-Neisseriacatarrhalis65+Micrococcuslysodeikticus35+Streptococcusfaecalis33+Staphylococcusaureus26+Bacillusmegaterium22+Saccharomycescerevisiae10treatmentwith95percentethanol.TheseresultsthussupporttheviewthatGrampositivityisduetothereducedaccessibilityoftheCV-Icomplextothesolvent,resultingprobablyfromareduc-tionintheporesizebydehydrationofwallmuco-complexesby95percentethanolandpossiblyalsofromthepresenceofthelargeiodineatom,whichmayofcoursebecomeassociatedwithpartsofthemoleculesasitdoesinotherpoly-saccharides(41).However,asiodineisaninhibi-z-__Uwenw-Jwc'J94[VOL.25 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